Abstract
We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA) and the transgene under the TET Responsive Element promoter (TRE) on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP) or turboFP635 (RFP) in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.
Highlights
The cerebral cortex of mammals consists of various types of projection neurons that are aligned in layers [1,2]
StTTrG was constructed by a series of plasmid cloning, by which a portion of STB [18] was PCRamplified to be cloned into the AscI-XbaI site of SARE-ArcMin lentiviral vector [19], replacing the SARE-d2EGFP with synapsinI-tTA, followed by cloning of MluI-ClaI fragment of TpGB into the BamHI-XhoI locus
We showed that StTTrG/fusion glycoprotein B type (FuG-B) vector can reveal the fine morphology of neuronal processes
Summary
The cerebral cortex of mammals consists of various types of projection neurons that are aligned in layers [1,2]. These projection neurons exhibit characteristic morphology and intrinsic connectivity, which lay basis for the canonical lamina circuit common across areas and species [3]. Much of our current knowledge on the morphology and connectivity of cortical projection neurons was obtained by variations of such single-cell analysis. A new generation of viral-based retrograde tracers is becoming a useful option to analyze the morphology of a defined set of projection neurons
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